CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice, we show that CAFs in both invasive lobular breast cancer and triple-negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo and in vitro studies reveal the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. Functional co-culture experiments show that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data suggest that CD26+ and CD26- NFs transform into distinct CAF subpopulations in mouse models of breast cancer.


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Any human data displayed in the manuscript (laser-microdissected tumor material from ILC and IDC patients, figure  The potential presence of significant outliers was determined using the Grubb's outlier test (Graphpad outlier calculator: https:// www.graphpad.com/quickcalcs/Grubbs1.cfm alpha: 0.05) . No significant outliers were identified in the data presented in this study.
All experiments within this study were performed for a minimum of three independent experiments. All in vivo transplantation experiments contained at least 5 animals per group/timepoint. The single cell transcriptomic experiments were performed with at least 2 animals per time point/group.
Randomization was applied where possible. Within the animal experiments limited randomization was applied as all animals needed to be of a certain genotype, age and sex. Mice transplanted with similar donor tissue were randomly allocated to the time point groups.
Our laboratory animal technicians were blinded to the treatments/groups of mice while assessing tumor volumetric measurements. Measurements of invasion within the organotypic invasions assays and cell counts of the transwell assays were performed blinded by two researchers and results were compared and concordant. Flow cytometry analysis of tumors from transplantation studies were not blinded since information regarding the tumor model was needed to ensure the correct FACS panel of antibodies was added to the samples. monocyte recruitment assays were done blinded (re-numbering of wells) and analyzed by FACS blinded. All remaining experiment were not done blinded as information regarding samples was essential to conduct the experiments in a proper way.  Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Methodology
Sample preparation Tumor cell lines used in this study were isolated from end-stage WEPtn-or WB1P-derived tumors. Normal fibroblasts (CD26-/ + NFs) were isolated by FACS from WapCre-negative Cdh1F/F;PtenF/F or mTmG (both FVB/n background) female mice between 8 and 16 weeks of age. Primary fibroblasts were kept in culture for no longer than passage 6 for experiments. Experiments were performed with the lowest passage number possible.
WEPtn and WB1P-derived tumor cell lines were tested for their EpCAM expression by flow cytometry to ensure pure tumor cell cultures without contamination from other cell types. Only cultures with >90% EpCAM cells were used for experiments. Furthermore, the tumor cell lines were genotyped to ensure deletion of target driver genes (loss of E-cadherin and Pten in WEPtn cells and loss of Brca1 and Trp53 in WB1P cells). Primary normal fibroblasts were analyzed for PDGFR-beta expression by flow cytometry and displayed spindle cell shaped morphology. No genotyping was performed on the fibroblasts.
All cell lines are regularly tested for mycoplasma in our lab and all cell lines and primary fibroblast cultures were tested negative for mycoplasma.
No misidentified cell lines were used in this study.
The following strains were used for this study: WapCre;Cdh1F/F;PtenF/F (WEPtn), WapCre;Cdh1F/F;Col1a1invCAG-Pik3caH1047R-IRES-Luc (WEH1047R), WapCre;Brca1F/F;P53F/F (WB1P) and WapCre;Brca1F/F;Trp53F/F;Col1a1invCAG-Myc-IRES-Luc (WB1P-Myc). All tumor models were on FVB/n background and generated in-house. mTmG reporter mice were backcrossed for seven generations to FVB/n background to accommodate transplantations with donor tissue from our FVB/n-based breast cancer mouse models. EN1-Cre mice were purchased from The Jackson Laboratory (JAX stock number:007916) and backcrossed with mTmG (FVB/n) mice for 2 generations to generate EN1-Cre;mTmG mice for in vivo tracing. All mice were housed on standard 12 hour day/night cycle in individually ventilated cages with ad libitum food. Room temperature was maintained at 21 degrees Celsius and humidity was 55%. The age of the experimental animals dependent on the experiments. In brief: for MMEC transplantations the donor mice were between 4 and 6 weeks old and the recipient mice were between 18-21 days old. For the bone marrow transplantations the recipients were 8 weeks old at time of irradiation. Bone marrow donors were age-and gender-matched. For the whole mammary gland transplantation the donors and recipients were 4 weeks old at time of transplantation. Details are in the material and methods section.
No wild animals were used in this study.
All experiments were performed with female mice, as this research focuses on breast cancer.
This study did not use field-collected samples.
All animal experiments were approved by the Dutch Animal Ethical Committee and conducted in compliance with the Netherlands Cancer Institute and Dutch Animal Welfare guidelines.
All tumors and control mammary glands were placed in PBS on ice upon harvesting. Samples were chopped into small pieces using a scalpel and processed into a single cell suspension using a digestion mix containing 2 mg/ml collagenase + 4ug/ml DNase in DMEM/F12. Samples were incubated for 60 minutes at 37°C under continuous shaking. After incubation the collagenase was inactivated by addition of equal volume of DMEM + 5% FCS. Samples were filtered through 70 um cell strainers and spun at 300g for 5 minutes to pellet the cells. Cell pellets were resuspended in red blood cell lysis buffer (RBC lysis, 155 mM NH4Cl, 10 mM KHCO3 anad 0.1 mM EDTA in H2O) and incubated on ice for 5 minutes. Next the samples were spun down, 300g for 5 minutes at 4°C. Cell pellets were resuspended in FACS buffer (1% BSA + 5 mM EDTA in PBS) and